Validation of Housekeeping Genes in the Brains of Rats Submitted to Chronic Intermittent Hypoxia, a Sleep Apnea Model

Abstract

Obstructive sleep apnea (OSA) is a syndrome characterized by intermittent nocturnal hypoxia, sleep fragmentation, hypercapnia and respiratory effort, and it has been associated with several complications, such as diabetes, hypertension and obesity. Quantitative real-time PCR has been performed in previous OSA-related studies; however, these studies were not validated using proper reference genes. We have examined the effects of chronic intermittent hypoxia (CIH), which is an experimental model mainly of cardiovascular consequences of OSA, on reference genes, including beta-actin, beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine guanine phosphoribosyl transferase and eukaryotic 18S rRNA, in different areas of the brain. All stability analyses were performed using the geNorm, Normfinder and BestKeeper software programs. With exception of the 18S rRNA, all of the evaluated genes were shown to be stable following CIH exposure. However, gene stability rankings were dependent on the area of the brain that was analyzed and varied according to the software that was used. This study demonstrated that CIH affects various brain structures differently. With the exception of the 18S rRNA, all of the tested genes are suitable for use as housekeeping genes in expression analyses.

Peer Review #1 of "Validation of endogenous reference genes in rat cerebral cortex for RT-qPCR analyses in developmental toxicity studies (v0.1)"

“…This is in contrast to previous studies testing across different rat tissues (Svingen et al, 2015) or brain tissue from rats (Cook et al, 2009;Yang et al, 2008;Zhou et al, 2016), mice (Crans et al, 2019) or humans (Rydbirk et al, 2016). Notably, brain tissue from a rat sleep apnea model displayed high stability of all tested RGs except 18S rRNA (Julian et al, 2014), a stability also seen in rat brains across fetal developmental stages (Al-Bader & Al-Sarraf, 2005), which agrees with our findings of relative high stability among RGs in this tissue (Julian et al, 2014). There are, however, studies showing unstable expression of common RGs depending on experimental parameters and what brain regions are examined (Cook et al, 2009;Swijsen et al, 2012;Yang et al, 2008;Zhou et al, 2016).…”

Lifestyle intervention in individuals with impaired glucose regulation affects Caveolin-1 expression and DNA methylation

“…We quantified the gene expression using the Comparative threshold (Ct) method (ΔΔCt method) [18,19], and the amount of target gene was normalized to ACTB as housekeeping gene (as it was stable across all samples) and determined by 2 −ΔΔCt , as previously described [20,21], with relative expression levels reported as fold change. Ct values higher than the cut-off of 35 were not considered as a reliable expression value, according to the manufacturer's recommendations, and therefore were excluded from the statistical analysis.…”

Validation of Reference Genes for qPCR Analysis of Resistance Training and Androgenic Anabolic Steroids on Hypothalamus, Adrenal Gland and Fat Tissue

“…This difference may be attributed to different mathematical models used in each program [24]. On the other hand, there was found similar results for the software geNorm and Normfinder but not for BestKeeper, this can be justified by the fact that this software used the Pearson correlation method to classify the reference genes, a different method compared to the others software [3].…”

“…Our group has validated reference genes for rat models of sleep deprivation [2] and hypoxia [3]. To our knowledge, there are no studies concerning validation of reference genes to analyze the effects of resistance exercise (REx) and androgenic anabolic steroids (AAS) use on gene expression.…”